Quadrupole ion trap mass spectrometers (QITMS) are used to provide rapid and sensitive analyses of a wide range of chemical and biochemical compounds. QITMSs and related spectrometric methods are known in the art and are as provided in U.S. Pat. No. 4,540,884, the entirety of which is incorporated herein by reference. Such instruments have begun to play a particularly important role in proteomics research as their favorable characteristics are applied to the identification, quantitation, and structural elucidation of peptides and proteins. One limitation with the QITMS, however, is that the structural analyses provided by this instrument is performed in a serial manner in the context of tandem mass spectrometry (MS/MS) experiments. With the emerging importance of proteomics the need for more rapid analyses are being realized.
A range of ions with different mass-to-charge (m/z) values can be trapped simultaneously in a quadrupole ion trap by the application of a radio frequency (rf) voltage to the ring electrode of the device. The trapped ions all oscillate at frequencies that are dependent on their m/z, and these frequencies can be readily calculated. MS/MS is then performed by carrying out three steps. First, the analyte ions having the single m/z of interest (parent ions) are isolated by changing the rf voltage applied to the ring electrode and by applying waveforms (i.e. appropriate ac voltages to the endcap electrodes) with the appropriate frequencies that resonantly eject all the ions but the m/z of interest. Second, the isolated parent ions are then resonantly excited via the application of another waveform that corresponds to the oscillation frequency of the parent ions. In this way, the parent ions' kinetic energies are increased, and they undergo energetic collisions with the background gas (helium), which ultimately result in their dissociation into product ions. Third, these product ions are then detected with the usual mass analysis techniques in QITMS. It is the mass differences between these product ions and their incipient parent ions that provides the structural information during this MS/MS experiment. This method of performing MS/MS is the current state-of-the art in commercial QITMS, and referred to as serial MS/MS.
Multiplexed MS/MS refers to performing MS/MS on ions of multiple m/z ratios simultaneously. A primary concern, however, is that upon isolation and dissociation of several compounds simultaneously, the product ions that are formed need to be associated with the correct parent ions in order for structural information to be gathered for each parent ion. During serial MS/MS this is accomplished by isolating and dissociating only one parent ion at a time so that the resulting products necessarily come from that parent ion. When one isolates and dissociates multiple parent ions all at once, the normal manner of relating which product ions dissociate from each parent ion is lost.
Several protocols for multiplexed MS/MS on Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometers have been reported. Comprehensive 2-dimensional (2-D) methods analogous to 2-D NMR were used to simultaneously dissociate a collection of parent ions. Attributing the resulting product ions to the appropriate parent ions relies on a sinusoidal pattern of excitation waveforms that produces a modulation in the product ion abundances that can be later deconvoluted. Hadamard transform methods have also been used, but like the comprehensive 2-D approach multiple spectra are acquired in which different subsets of parent ions are simultaneously dissociated. The drawback to both the Hadamard method and the comprehensive 2-D approach is that these methods provide little to no timesavings as compared to the analogous serial approaches. Further, parent ion dissociations and product ion abundances do not always vary in the expected manner. Encoding is dependent upon known changes in parent ion kinetic energy, but product ion abundances do not necessarily change in a direct manner as parent ion kinetic energies (and, thus, collision energies) are changed. The net result is that product ions may be associated with incorrect parent ions.
Another approach developed recently allows product ion spectra to be obtained from multiple parent ions in a single mass spectrum, which significantly enhances the throughput. Because MS/MS analyses on FTICR mass spectrometers are inherently slower than MS/MS analyses on QITMS, this method is noticeably slower. Furthermore, this approach relies on the high mass accuracy of the FTICR to identify product ions from different parent ions by exact mass and database searching. Consequently, this method necessitates the high performance capabilities offered only by FTICR spectrometers, and therefore is not suitable for cheaper and more widely accessible mass spectrometers like QITMS. Further, this method depends upon the compound of interest present in an accessible data base and effective search capabilities—without which the analysis is unworkable.